Improved strategy for recombinant production and purification of antimicrobial peptide tachyplesin I and its analogs with high cell selectivity

Abstract

Here, we report an efficient procedure for recombinant production and purification of tachyplesin I (THI) with a final yield of 17 mg/L of the culture medium. The peptide was expressed in Escherichia coli as a part of the thioredoxin fusion protein. With the use of soluble expression followed by immobilized metal-ion affinity chromatography, the recombinant protein cleavage and reversed-phase high-performance liquid chromatography, a yield of THI did not exceed 6.5 mg/L of the culture medium. Further optimization studies were carried out to improve the protein expression level and simplify purification procedure of the target peptide. To achieve better yield of the peptide, we used high-cell-density bacterial expression. The formed inclusion bodies were highly enriched with the fusion protein, which allowed us to perform direct chemical cleavage of the inclusion bodies solubilized in 6 M guanidine-HCl with subsequent selective precipitation of proteins with trifluoroacetic acid. This enabled us to avoid an extra step of purification by immobilized metal-ion affinity chromatography. The developed procedure has made it possible to obtain biologically active THI and was used for screening a number of its mutant analogs. As a result, several selective and nonhemolytic analogs were developed. Significant reduction in hemolytic activity without losing antimicrobial activity was achieved by substitution of tyrosine or isoleucine residue in the β-turn region of the molecule with hydrophilic serine. The present study affords further insight into molecular mechanism of antimicrobial action of tachyplesin and gains a better understanding of structure-activity relationships in its analogs. This is aimed at searching for novel antibiotics on the basis of antimicrobial peptides with reduced cytotoxicity.

Effect of N- and C-Terminal Modifications on Cytotoxic Properties of Antimicrobial Peptide Tachyplesin I

“…Recombinant tachyplesin I without N-or C-terminal modifications was expressed in Е. coli [9]. The calculated molecular weight of nonmodified and N-and C-terminally modified tachyplesins were 2264.10 and 2305.10 Da, respectively.…”

“…It has been shown that tachyplesin I possessed a cytotoxic effect on HL-60 cells (acute human promyelocytic leukemia cells) [11], K562 cells (myelogenous leukemia cells) [10], TSU cells (prostate cancer cells) [1]. The first two studies focused on antitumor activity of natural tachyplesin I with amidated C-terminal residue, in the third report, tachyplesin I with modifications at both termini was used.We have recently developed a novel approach to producing recombinant tachyplesin I analogs [9]. Despite the absence of post-translational C-terminal amidation, the recombinant peptide exhibits both antibacterial and antitumor activities.…”

“…We have recently developed a novel approach to producing recombinant tachyplesin I analogs [9]. Despite the absence of post-translational C-terminal amidation, the recombinant peptide exhibits both antibacterial and antitumor activities.…”

Analysis of Synergistic Effects of Antimicrobial Peptide Arenicin-1 and Conventional Antibiotics

“…Recombinant arenicin-1 and tachyplezin-1 were obtained by heterologous expression in E. coli cells [9,10]. For antibiotic activity assessment, bacterial strains E. coli C600, S. aureus 209P and P. aeruginosa PAO1 were used as test cultures.…”

Recombinant expression of LFchimera antimicrobial peptide in a plant-based expression system and its antimicrobial activity against clinical and phytopathogenic bacteria

“…In another study, the recombinant MAP30 antimicrobial protein was effectively expressed and targeted to ER in Nicotiana tabacum hairy roots; it exhibited significant antibacterial activity [73], and ER-signal was used for successful expression of a plant defencin protein in transgenic tobacco seeds [53]. However, because AMPs have natural destructive effects on microorganisms and are relatively sensitive to proteolytic degradation, they are often produced by fusion to a partner in heterologous hosts and/or by modifying different parameters during fermentation to obtain successful recombinant production [74][75][76].…”